We seek to understand, at the cell biological and molecular levels, how chromosomes move, how they are guided to the metaphase plate, and how microtubule interactions with the kinetochore and other mitotic organelles regulate the mitotic spindle checkpoint. Chromosome movements during prometaphase are driven primarily by dynamic interactions of the mitotic spindle microtubules with the kinetochores. At the same time, kinetochores lacking stable bipolar attachment to microtubules serve to signal the spindle checkpoint that blocks anaphase onset until metaphase alignment is achieved. This checkpoint is silenced when the all sister kinetochores establish stable bipolar microtubule attachments. Thus, chromosome attachment and movement on the mitotic spindle are intimately intertwined with the regulation of the spindle checkpoint. Previously, this lab provided evidence that translocation of the kinetochores along microtubules is the prime mediator of chromosome movement in mitosis. We later discovered that individual kinetochores within a mitotic cell were biochemically distinct and developed the model of kinetochores as catalytic sources for spindle checkpoint signaling. We have now assembled a unique set of tools to dissect the molecular roles of several of the most important regulators of chromosome movement and the spindle checkpoint in vertebrate cells. We will determine if and how microtubule attachment and mechanical tension regulate kinetochore protein dynamics and signaling. We focus on a set of key regulators of microtubule-kinetochore interactions: the Ndc80 protein complex, Polo-like kinase1, the Aurora B kinase, the dynein/dynactin complex and an associated protein complex, the ZW10/Rod complex. We will analyze the functions of these proteins in living Xenopus and mammalian cells. We will recapitulate kinetochore regulation in lysed cell systems, in fractions from mitotic cells, and with purified proteins in vitro. We will collaborate to conduct complementary studies in Xenopus egg extracts. Analyzing the roles of several dynamic protein complexes is a significant challenge. However, only by approaching kinetochore and microtubule protein dynamics in concert with a variety of in vivo and in vitro approaches can we begin to understand the regulation of mitosis in living cells. [unreadable] [unreadable]